The trypanosomes and leishmania are parasitic protozoa that cause African sleeping sickness, Chagas' disease and leishmaniasis. Together, these diseases afflict millions of people, and can be lethal if not treated. The available therapeutic agents are wholly unsatisfactory. The long-term objective of this work is to assess whether the type II topoisomerases, a fascinating, and relatively newly-discovered class of enzymes are a suitable target for new anti-trypanosomal chemotherapy, as suggested by pilot studies. This project will document the effects of potent, and highly specific inhibitors of type II topoisomerases (such as the anti-tumor agent, VP16-213) on the structure and replication of DNA in Trypanosoma equiperdum, and will correlate these effects with anti-trypanosomal activity in mice. The possibility that classical anti-trypanosomal drugs (ethidium bromide, pentamidine and other diamidines) have as their mechanism of action the inhibition of topoisomerases will be explored. VP16-213 and anti-trypanosomal drugs will be tested against purified mitochondrial and nuclear topoisomerases from T. equiperdum. Polyamines will be assessed as possible antagonists of the molecular and therapeutic effects of topoisomerase inhibitors, and alpha-difluoromethylornithine (an inhibitor of polyamine biosynthesis in trypanosomes) will be evaluated for possible synergy with topoisomerase inhibitors. In an effort to determine whether the nuclear and mitochondrial type II topoisomerases are different, drug-sensitive topoisomerase(s) will be isolated from nuclear and mitochondrial protein-DNA complexes, and compared with enzymes purified from nuclei and mitochondria of T. equiperdum. Evidence for a maxi- circle-encoded type II topoisomerase gene will be sought; if found, the cDNA will be sequenced and compared with that of the nuclear gene. Possible topological determinants of the drug-promoted cleavage patterns seen in vivo will be sought, using purified mitochondrial enzyme and various topological forms of minicircle DNA. These studies will expand our understanding of the role of topoisomerases in the maintenance and replication of DNA in trypanosomes. It is hoped that they will also provide a rational basis for much-needed new anti-trypanosomal chemotherapy.